Use of substance P antagonists in the treatment of the adenocarcinomas

ABSTRACT

It is described the use of antagonists of neuro kynin receptors (NK-1; substance P receptors) in order to establish a new drug effective in the treatment of adenocarcinomas, such antagonists being one or more substances selected among those ones having the following features: pA 2 &gt;6.0 both in human and in murine tissues, etherocyclic non peptidergic structures, antiangiogenic effects experimentally demonstrated onto the genito-urinary tract rumors induced via orthotopic drafts of human tumoral cells either in the genito-urinary apparatus of either immunodeficient rats or mice, decrease of the tumoral mass on tumors of the genito-urinary tract induced by orthotopic drafts of tumoral cells onto tissues of the genito-urinary tract of either immunodeficient rats or mice.

FIELD OF INVENTION

The present invention refers to, the use of substance P antagonistsparticularly the invention refers to the use of NK-1 receptorantagonists for the treatment of adenocarcinomas, more particularlygenito-urinary-tract neoplasms, more particularly prostatic carcinoma.

STATE OF THE ART

Malignant neoplasms, originating from epithelial cells are namedcarcinomas. A peculiar type of carcinoma, of glandular origin, is theadenocarcinomas. Since a correlation between tumors and angiogenesis hasbeen hypothesized, a possible, effective strategy against cancer diseaseis a pharmacological treatment with angiogenesis inhibitors.

Angiogenesis, i.e. the formation and development of new capillaryvessels, occurs in different physiologic conditions, such as embryonaldevelopment. On the other hand, intense angiogenesis occurs in severalpathologic conditions, such as synovial rheumatoid hypertrophy,atherosclerosis, proliferative retinopathy and solid tumors. Withreference to solid tumors, the interest in the neovascularisation hasbeen raised from the evidence that tumors cannot growth or metastasizewithout new vessels and/or growth factors. Solid tumors cannot growbeyond 1-2 mm³ without neovascularation, which furnishes feeding totumors. Therefore, experiments have been carried out in order toquantify neovascularation in order to try to evaluate the tumor growthat different stages [Tosan A., Fregene et al, Anticancer Research 13:2377-2382 (1993); 13: Brigitte V. Offersen et al., APMIS 106: 463-469(1998)]. Consequently, it has been hypothesized that tumor growth couldbe prevented by neovascularization blockage.

Experimental evidences have recently outlined that substance P (SP)plays a role in angiogenesis stimulation:

Daily administration of substance P causes intense neovascularization ina rat sponge model of angiogenesis [T.-P. D. Fan et al, Br. J.Pharmacol. 110: 43-49 (1993)];

The angiogenic response towards SP can be blocked by using selectiveantagonists for NK-1 receptors for tachykinines [T.-P. D. Fan et al, Br.J. Pharmacol. 110: 43-49 (1993)];

The angiogenic activity of SP can be counteracted by administration ofeither peptide or non peptide antagonists [D. Regoli et al., Pharmacol.Rev., Vol. 64, No. 4 551-559 (1994)].

EP 0835662 describes peptide antagonists of substance P which arecharacterized by negative side effects. Henning Ivo M. et al., Int. J.Cancer. 61, 786-792 (1995) describes binding experiments for substance Preceptors and hypothesizes that the progression of tumor is mediated viaangiogenetic mechanisms.

Such experimental results have not yet led to identify any drug whichcould be successfully employed in the treatment of the adenocarcinomas,particularly the prostatic carcinoma. As a matter of fact, Fan et al.states about future therapeutic applications in the above mentionedmechanism.

Moreover, NK-1 antagonists show a great variability in their molecularstructure (Regoli et al, 1994), therefore being very difficult topredict potential activity based upon their structure-activityrelationship. Also, insofar, no data have currently shown that theantagonistic activity and selectivity towards the human subtype NK-1receptor could be of interest in the therapy of adenocarcinoma,particularly of the prostatic adenocarcinoma. In relation to the currentstate of the art regarding cancer therapy, none of the tested substancesseems to be effective in cancer therapy. Therefore for the substancesmentioned in Regoli it cannot be inferred any specific activity againstcancer.

With the present invention, we aim at inhibiting, via administration ofinhibitors of angiogenesis and particularly by using of antagonists ofNK-1 tachykinergic receptor, solid tumor growth specifically localizedto the genito-urinary tract. This innovative methodology eithersubstitutes or integrates current therapies against cancer, such assurgery, chemiotherapy and radiotherapy.

SUMMARY OF THE INVENTION

It is an object of the present invention the use of NK-1 receptorantagonists in the treatment of the adenocarcinomas, particularly in thetreatment of the carcinomas of the genito-urinary tract and moreparticularly in the treatment of the prostatic adenocarcinoma.

Another object of the invention is the use of substance P antagonists inthe treatment of the adenocarcinomas, particularly in the treatment ofthe carcinomas of the genito-urinary tract and more particularly in thetreatment of the prostatic adenocarcinoma.

Further objects of the invention will be evident by the followingdetailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to the use of substance P receptorantagonists particularly NK-1 receptor antagonists, in the treatment ofadenocarcinomas, particularly genito-urinary tract neoplasms, moreparticularly prostatic carcinoma. Selected antagonists according to thepresent invention are listed in Tab. 1 and are characterised by thefollowing features:

1) pA₂>6.0 both in murine and human tissues, pA₂ being the concentrationof the drug at which it is half-maximally effective, and the pA₂ isdirectly related to the affinity of the ligand to the receptor,

2) Non peptidic heterocyclic structure.

3) Antiangiogenic effects experimentally evaluated onto tumors of thegenito-urinary tract induced via orthotopic grafts of tumoral humancells on tissues of the genito-urinary tract of rats and/orimmunodeficient mice. Orthotopic graft is intended to be a graft ofcells in the host, via direct injection of cells.

4) Reduction of the tumoral mass experimentally demonstrated onto tumorsof the genito-urinary tract induced via orthotopic grafts of tumoralhuman cells on tissues of the genito-urinary tract of rats and/orimmunodeficient mice.

The following substances are considered by the authors effective againstadenocarcinomas, specifically against the adenocarcinomas originatingfrom the genito-urinary tract.

TABLE 1 pA₂ pA₂ Compound human rat References FK 888 9.1 6.0 Fujii etal., Br. J. Pharm. 107:785, 1992 CP 96345 9.5 6.8 Srider et al., Science251:435, 1991 CP 99994 8.9 6.1 Desal et al., J. Med. Chem. 35:4911, 1992SR 140333 9.8 7.4 Edmonts et al., Eur. J. Pharm. 250:403, 1993 CGP47899 >6.0 >6.0 Shilling et al., Pers. Med. Chem. 207, 1993 RP 67580 7.28.2 Garret et al., PNAS 88:10208, 1991 MEN 11149 >6.0 >6.0 Cirillo etal., Eur. J. Pharm. 341:201, 1998 MEN 11467 >6.0 >6.0 Evangelista etal., XXIX Nat. Congr. of the Ital. Pharmacological Soc., Florence20-23.06, 1999 GR 205171 >6.0 >6.0 Gardner et al., Regul Pep. 65:45,1996 L-703,606 Cascieri et al., Mol. Pharmacol. 42, 458, 1992

The above mentioned substances may be used to prepare drugs incombination with known adjuvants. Sinergistically, they can be combinedwith substances listed in table 2. As a matter of fact, the combinationof one or more substances listed in table 1 with one or more substancesin table 2 (or corresponding derivatives of the substances listed intable 2, derivatives known to the man skilled in the art) provides atherapeutic positive response higher than 10%, as stated in points 3-4of the pharmacological characteristics of the NK-1 antagonists.

TABLE 2 FLUTAMIDE (Eulexin by Schering; Drogenil Preferred dose range:by Essex). 15-1500 mg/day LEUPROLIDE ACETATE (Enantone by Preferred doserange: Takeda). 0.1-10 mg/month GOSERELIN (Zoladex by Zeneca). Preferreddose range: 0.1-10 mg/28 days AMINOGLUTETHIMIDE (Orimeten by Preferreddose range: Ciba-Geigy). 30-3000 mg/day KETOKONAZOLE (Zinoral byJanssen). Preferred dose range: 10-1000 mg/day DOXORUBICINA(Adriblastina by Pharmacia Preferred dose range: & Upjohn). 2-100 mg/dayTAXOL Preferred dose range: 2-100 mg/day

Route of administration is based upon the specific characteristics ofthe compounds and implies the endovenous, intrabladder, intraperitoneal,intramuscular, subcutaneous and oral administration.

Adjuvants are selected among those commonly used in pharmacotherapy,such as: methyl-p-hydroxybenzoate, latex and saline solution.

Administration of substances listed in table 1, possibly in combinationwith substances listed in Table 2, is able to either reduce or reversetumor growth via inhibition of angiogenesis and tumoral mass.

Moreover, use of antagonists of substance P is endowed of the followingadvantages:

Antiemetic effect, opposite to chemiotherapic drugs which show markedemetic effects

Antidepressive effect, which is a very important psychotherapic effectin cancer affected patients.

The following examples should be considered as illustrative of thepresent invention an not limitative of the scope of the inventionitself.

MATERIALS AND METHODS Animals

For this studies has been used male athymic nude mices (Harlan). Themices were housed in laminar flow cabinet under pathogenic freeconditions and used at 4-5 weeks of age.

Orthotopic Implantation

The PC-3 human prostatic cancer cell lines (ECACC) were mantained inMinimum essential medium (GIBCO BRL) For in vivo studies, tumor cells inexponential growth phase were harvested by a 120 seconds treatment withtripsyn in 0.02% EDTA. After that the cells were resuspended in salinesolution. 20 μl (10⁵ cells) of saline solution was inoculated in nudemice prostate. The tumors were taken at different times (three up to 180days) for analysis.

Treatments

Animals were divided in groups (10 animals each one) and treated withdifferent solutions:

physiological solution, -Sustance P (1 ng up to 1 mg), -L-703,606 (1 ngup to 1 mg), −L-703,606+Leuprolide Acetate (using dosages reported intable 1 and 2).

The treatments were made at different time (one at day until one atweek). For different periods of time (three days up to 180 days).

Immunohystochemistry

Frozen sections of tumors have been fixed in Acetone,acetone/chloroform, acetone.

The presence of new blood vessels has been highlighted using the Ratanti-Mouse CD31 antibody, a Goat anti-Rat POX as a secondary antibodyand DAB as a chromogen substrate. For quantification of blood vessels animage analysis system was employed.

Cancerogenicity

The animals were maitained under observation for three up to 180 days.Autoptic examinations were performed in all animals and the tumoral massweighed.

Results

Vascular Density Quantification

The quantitative data obtained on blood vessels density have shown astatistically significant difference (p<0.05) amongst differenttreatments. SP treated mice group showed a statistically significantincrease of CD 31 values compared to control groups (p<0.05).

Moreover, a statistically significant reduction (p<0.05) of CD 31 valueshas been outlined in mice treated with L-703,606 compared either tocontrol groups or SP treated mice. The inhibitory angiogenic effectmediated by L-703,606 has been shown in all groups treated with thiscompound. A statistically significant reduction of CD 31 values has beenshown with L-703,606+Leuprolide, Acetate (p<0.05) compared to L-703,606groups (>10%).

Tumoral Mass Weight

Tumoral mass weight has been evaluated in all experimental groups,throughout treatment days (from 3 up to 180 days). Results have shown astatistically significant reduction (p<0.05) of tumoral mass weight inmice treated with L-703,606, compared to either control groups or SPtreated mice. Moreover, combination of L-703,606 with Leuprolide Acetateallows to reveal a further tumoral mass reduction, (>100%).

Correlation between tumoral mass weight and type of treatment isproportionally related to treatment time-course.

What is claimed is:
 1. A method for treating adenocarcinomas comprisingthe steps of administering to a subject in need of treatment apharmaceutically effective amount of an antagonists of NK-1 receptors,such antagonists being one or more compounds and corresponding mixturesselected among the compounds having the following characteristics:pA₂>6.0 in human and murine tissue; non peptidic heterocyclic structure;antiangiogenetic effects experimentally demonstrated in urogenitaltumors, induced by orthotopic implantation of human tumoral cells inimmunodeficient rat and mice urogenital tissues, said orthotopicimplantation being made by inoculating the rat and mice urogenitaltissues with said human tumoral cells and said angiogenetig effectsbeing measured by the reduction of tumoral mass in the urogenital tumorsinduced by the orthotopic implantation.
 2. The method according to claim1 wherein the compounds are selected among: FK 888, CP 96345, CP 99994,SR 140333, CGP 47899, RP 67580, MEN 11149, MEN 11467, GR 205171,L-703,606 and corresponding derivatives and corresponding mixtures. 3.The method according to claim 1 wherein the compound is combined with atleast one compound selected among: FLUTAMIDE, LEUPROLIDE, GOSERELIN,AMINOGLUTETHIMIDE, KETOKONAZOLE, DOXORUBICINA, TAXOL and correspondingderivatives and corresponding mixtures.
 4. The method according to claim3 wherein each of the compounds is administered at the following dosage:FLUTAMIDE 15-1500 mg/day, LEUPROLIDE ACETATE 0.1-10 mg/month, GOSERELIN0.1-10 mg/28 days, 30-3000 mg/day, 10-1000 mg/day, DOXORUBICINA 2-100mg/day, TAXOL 2-100 mg/day.
 5. The method according to claim 1 whereinthe compounds are administered via endovenous, intrabladder,intraperitoneal, intramuscular, subcutaneous and oral route.
 6. Themethod according to claim 1 wherein the compounds are mixed withadjuvants selected in the group consisting of: metil-p-hydroxybenzoate,latex, saline solution and corresponding mixtures.
 7. The methodaccording to claim 1, wherein the adenocarcinomas are urogenitaladenocarcinomas.
 8. The method according to claim 1, wherein theadenocarcinomas are prostatic adenocarcinomas.
 9. A method to induce andtest effects of NK-1 receptor antagonists in immunodeficient rat andmice urogenital tissues comprising the steps of: (i) making anorthotopic implantation by inoculating the mice and rat urogenitaltissues with human tumoral cells, (ii) treating the tumoral cells afterimplantation with at least a compound, having pA₂>6.0 in human andmurine tissue and non peptidic heterocyclic structure, for a periodranging between 3 to 180 days, and (iii) testing on frozen sections oftumors of the rat and mice the effect of the compound by measuring thetumoral mass reduction, said frozen section being taken where theinoculation has been made.
 10. The method according to claim 9 whereinthe human prostatic cells are the PC-3 human prostatic cancer celllines.
 11. The method according to claim 9 wherein the effect of thecompound is tested by using Rat anti-Mouse CD31 antibody, Goat anti-RatPOX as a secondary antibody and DAB as a chromogen substrate.